M. Köller

The influence of proteins on the dispersability and cell-biological activity of silver nanoparticles

Spherical silver nanoparticles with a diameter of 50 ± 20 nm and stabilized with either poly(N-vinylpyrrolidone) (PVP) or citrate were dispersed in different cell culture media: (i) pure RPMI, (ii) RPMI containing up to 10% of bovine serum albumin (BSA), and (iii) RPMI containing up to 10% of fetal calf serum (FCS). The agglomeration behavior of the nanoparticles was studied with dynamic light scattering and optical microscopy of individually tracked single particles. Whereas strong agglomeration was observed in pure RPMI and in the RPMI–BSA mixture within a few hours, the particles remained well dispersed in RPMI–FCS. In addition, the biological effect of PVP-stabilized silver nanoparticles and of silver ions on human mesenchymal stem cells (hMSCs) was studied in pure RPMI and also in RPMI–BSA and RPMI–FCS mixtures, respectively. Both proteins considerably increased the cell viability in the presence of silver ions and as well as silver nanoparticles, indicating a binding of silver by these proteins.

Total parenteral nutrition with glutamine dipeptide after major abdominal surgery - A randomized, double-blind, controlled study

OBJECTIVE To assess the efficacy of glutamine (Gln) dipeptide-enriched total parenteral nutrition (TPN) on selected metabolic, immunologic, and clinical variables in surgical patients. SUMMARY BACKGROUND DATA Depletion of Gln stores might lead to severe clinical complications. Recent studies indicate that the parenteral provision of Gln or Gln-containing dipeptides improves nitrogen balance, maintains the intracellular Gln pool, preserves intestinal permeability and absorption, and shortens hospital stay. METHODS Twenty-eight patients (age range, 42-86 years, mean 68 years) undergoing elective abdominal surgery were allocated, after randomization, to two groups to receive isonitrogenous (0.24 g nitrogen kg(-1) day(-1)) and isoenergetic (29 kcal/122 kJ kg(-1) day(-1)) TPN over 5 days. Controls received 1.5 g of amino acids kg(-1) day(-1), and the test group received 1.2 g of amino acids and 0.3 g of L-alanyl-L-glutamine (Ala-Gln) kg(-1) day(-1). Venous heparinized blood samples were obtained before surgery and on days 1, 3, and 6 after surgery for routine clinical chemistry and for the measurement of plasma free amino acids. Lymphocytes were counted and the generation of cysteinyl-leukotrienes from polymorphonuclear neutrophil granulocytes was analyzed before surgery and on days 1 and 6 after surgery. Nitrogen balances were calculated postoperatively on days 2, 3, 4, and 5. RESULTS No side effects or complaints were noted. Patients receiving Gln dipeptide revealed improved nitrogen balances (cumulative balance over 5 days: -7.9 +/- 3.6 vs. -23.0 +/- 2.6 g nitrogen), improved lymphocyte recovery on day 6 (2.41 +/- 0.27 vs. 1.52 +/- 0.17 lymphocytes/nL) and improved generation of cysteinyl-leukotrienes from polymorphonuclear neutrophil granulocytes (25.7 +/- 4.89 vs. 5.03 +/- 3.11 ng/mL). Postoperative hospital stay was 6.2 days shorter in the dipeptide-supplemented group. CONCLUSION We confirm the beneficial effects of Gln dipeptide-supplemented TPN on nitrogen economy, maintenance of plasma Gln concentration, lymphocyte recovery, cysteinyl-leukotriene generation, and shortened hospital stay in surgical patients.

Uptake and intracellular distribution of silver nanoparticles in human mesenchymal stem cells

Silver nanoparticles (Ag-NP) are widely used due to their well-known antibacterial effects. In medicine Ag-NP have found applications as wound dressings, surgical instruments and bone substitute biomaterials, e.g. silver-containing calcium phosphate cements. Depending on the coating technique, during resorption of a biomaterial Ag-NP may come into close contact with body tissues, including human mesenchymal stem cells (hMSC). Despite the widespread uses of Ag-NP, there is a serious lack of information concerning their biological effects on human cells. In this study the uptake of Ag-NP into hMSC has been analyzed and the intracellular distribution of Ag-NP after exposure determined. Non-agglomerated (dispersed) Ag-NP from the cell culture medium were detected as agglomerates of nanoparticles within the hMSC by combined focused ion beam/scanning electron microscopy. The silver agglomerates were typically located in the perinuclear region, as determined by light microscopy. Specific staining of cellular structures (endo-lysosomes, nuclei, Golgi complex and endoplasmatic reticulum) using fluorescent probes showed that the silver nanoparticles occurred mainly within endo-lysosomal structures, not in the cell nucleus, endoplasmic reticulum or Golgi complex. Quantitative determination of the uptake of Ag-NP by flow cytometry (scattergram analysis) revealed a concentration-dependent uptake of the particles which was significantly inhibited by chlorpromazine and wortmannin but not by nystatin, indicating clathrin-dependent endocytosis and macropinocytosis as the primary uptake mechanisms.

Influence of a total parenteral nutrition enriched with omega-3 fatty acids on leukotriene synthesis of peripheral leukocytes and systemic cytokine levels in patients with major surgery.

OBJECTIVE Nutritive immunomodulation of patients after major surgery. DESIGN Prospective, randomized controlled double-blind study. PATIENTS Forty patients undergoing major intestinal surgery. Patients were divided into two groups: one received a total parenteral nutrition enriched with omega-3 fatty acids for 5 days postoperatively, the other an isocaloric, control nutrition. MATERIALS AND METHODS We analyzed the leukocyte ability to release leukotrienes from whole blood leukocytes stimulated with the calcium ionophore A23187 (5 micromol/L) by reverse phase high-performance liquid chromatography and circulating cytokines by enzyme-linked immunosorbent assay. RESULTS Leukocytes from patients of the omega-3 fatty acids group generated significantly higher amounts of less biologically active leukotriene B5 as compared to the control group (p < or = 0.001). This was accompanied by a significant decrease in the generation of proinflammatory leukotriene B4 (p < or = 0.006) in the study group. In contrast to interleukin-1beta, interleukin-6, and interleukin-10 systemic levels of tumor necrosis factor-alpha (p < or = 0.05) were postoperatively decreased in the study group. CONCLUSION Our data provide evidence that a total parenteral nutrition enriched with omega-3 fatty acids modulates the lipid mediator pattern and systemic tumor necrosis factor-alpha levels.

The dissolution and biological effects of silver nanoparticles in biological media

Silver ions and silver nanoparticles have a well-known biological effect that typically occurs in biological or environmental media of complex composition. Silver nanoparticles release silver ions if oxidizing species like molecular oxygen or hydrogen peroxide are present. The presence of glucose as a model for reducing sugars has only a small effect on the dissolution rate. In the presence of chloride ions, precipitation of silver chloride nanoparticles occurs. At physiological salt concentrations, no precipitation of silver phosphate occurs as the precipitation of silver chloride always occurs first. If the surface of a silver nanoparticle is passivated by cysteine, the dissolution is quantitatively inhibited. Upon immersion of silver nanoparticles in pure water for 8 months, leading to about 50% dissolution, no change in the surface was observed by transmission electron microscopy. A model for the dissolution was derived from immersion and dissolution experiments in different media and from high-resolution transmission electron microscopy. A literature survey on the available data on the dissolution of silver nanoparticles showed that only qualitative trends can be identified as the nature of the nanoparticles and of the immersion medium are practically never comparable. The dissolution effects were confirmed by cell culture experiments (human mesenchymal stem cells and neutrophil granulocytes) where silver nanoparticles that were stored under argon had a clearly lower cytotoxicity than those stored under air. They also led to a less formation of reactive oxygen species (ROS). This underscores that silver ions are the toxic species.

Serum CD14 levels in poly traumatized and severely burned patients

Recently it has been demonstrated that the CD14 molecule which is expressed on monocytes and macrophages serves as a receptor for lipopolysaccharide (LPS) bound to LPS‐binding protein (LBP) and thus mediates LPS‐induced tumour necrosis factor (TNF) production. Here we report that CD14 is found as a soluble (s) molecule in serum. In healthy volunteers sCD14 levels (mean ± s.e.m.) were 3.7±0.05 μg/ml (n= 30, 25–50 years of age) as determined by ELISA (detection limit 20 μg/ml serum) using two monoclonal antibodies in a sandwich technique. In polytraumatized patients (n= 16) significantly decreased levels (1.7 ± 0.3) were detected immediately after the trauma, which increased to 4.9±0.3 μg/ml within the first 6 days post trauma. sCD14 remained elevated during the first 14 days post trauma in patients with the most severe injuries (injury severity score > 45 points), whereas a return to normal levels was observed in patients with an injury score of < 45 points. In addition, the levels of the high‐density lipoproteins that partially inactivate free endotoxin are significantly decreased post trauma. No correlation between parameters of inflammation (C3a and neopterin levels, leucocyte counts, amount of band cells), liver function and sCD14 levels was established. Comparable to polytraumatized patients, increased sCD14 serum levels were observed in five patients with burn trauma (burned area > 35%) within the second week post trauma when clinical signs of septicaemia were evident.

The toxic effect of silver ions and silver nanoparticles towards bacteria and human cells occurs in the same concentration range

Silver is commonly used both in ionic form and in nanoparticulate form as a bactericidal agent. This is generally ascribed to a higher toxicity towards prokaryotic cells than towards mammalian cells. Comparative studies with both silver ions (such as silver acetate) and polyvinylpyrrolidone (PVP)-stabilized silver nanoparticles (70 nm) showed that the toxic effect of silver occurs in a similar concentration range for Escherichia coli, Staphylococcus aureus, human mesenchymal stem cells (hMSCs), and peripheral blood mononuclear cells (PBMCs), i.e. 0.5 to 5 ppm for silver ions and 12.5 to 50 ppm for silver nanoparticles. For a better comparison, bacteria were cultivated both in Lysogeny broth medium (LB) and in Roswell Park Memorial Institute medium (RPMI)/10% fetal calf serum (FCS) medium, as the state of silver ions and silver nanoparticles may be different due to the presence of salts, and biomolecules like proteins. The effective toxic concentration of silver towards bacteria and human cells is almost the same.

Linezolid Penetration into Bone and Joint Tissues Infected with Methicillin-Resistant Staphylococci

ABSTRACT Penetration of linezolid into bone and joint tissues was studied by high-performance liquid chromatography in 13 patients suffering from implant-associated infections with methicillin-resistant staphylococci. Mean concentrations of linezolid in infected tissues were greater than 10 mg/liter in a sampling time range of 35 to 124 min after administration of the preoperative dose, except in bone specimens, where they reached 3.9 ± 2.0 mg/liter.

Easy assessment of the biocompatibility of Ni-Ti alloys by in vitro cell culture experiments on a functionally graded Ni-NiTi-Ti material.

The biocompatibility of nickel-titanium alloys was investigated by single-culture experiments on functionally graded samples with a stepwise change in composition from pure nickel to pure titanium, including an Ni-Ti shape memory alloy for a 50:50 mixture. This approach permitted a considerable decrease of experimental resources by simultaneously studying a full variation of composition. The results indicate a good biocompatibility for a nickel content up to about 50%. The cells used in the biocompatibility studies comprised osteoblast-like osteosarcoma cells (SAOS-2, MG-63), primary human osteoblasts (HOB), and murine fibroblasts (3T3).

Cell type-specific responses of peripheral blood mononuclear cells to silver nanoparticles

Silver nanoparticles (Ag-NP) are increasingly used in biomedical applications because of their remarkable antimicrobial activity. In biomedicine, Ag-NP are coated onto or embedded in wound dressings, surgical instruments and bone substitute biomaterials, such as silver-containing calcium phosphate cements. Free Ag-NP and silver ions are released from these coatings or after the degradation of a biomaterial, and may come into close contact with blood cells. Despite the widespread use of Ag-NP as an antimicrobial agent, there is a serious lack of information on the biological effects of Ag-NP on human blood cells. In this study, the uptake of Ag-NP by peripheral monocytes and lymphocytes (T-cells) was analyzed, and the influence of nanosilver on cell biological functions (proliferation, the expression of adhesion molecules, cytokine release and the generation of reactive oxygen species) was studied. After cell culture in the presence of monodispersed Ag-NP (5-30μgml(-1) silver concentration), agglomerates of nanoparticles were detected within monocytes (CD14+) but not in T-cells (CD3+) by light microscopy, flow cytometry and combined focused ion beam/scanning electron microscopy. The uptake rate of nanoparticles was concentration dependent, and the silver agglomerates were typically found in the cytoplasm. Furthermore, a concentration-dependent activation (e.g. an increased expression of adhesion molecule CD54) of monocytes at Ag-NP concentrations of 10-15μgml(-1) was observed, and cytotoxicity of Ag-NP-treated monocytes was observed at Ag-NP levels of 25μgml(-1) and higher. However, no modulation of T-cell proliferation was observed in the presence of Ag-NP. Taken together, our results provide the first evidence for a cell-type-specific uptake of Ag-NP by peripheral blood mononuclear cells (PBMC) and the resultant cellular responses after exposure.